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1、2019/6/30,1,Welcome to My Molecular Biology Class,Yang Yang（杨 洋 教授） Huazhong University of Sciences and Technology,2019/6/30,2,Molecular Biology of the Gene, 6/E - Watson et al. (2008),Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regul
2、ation Part V: Methods,2019/6/30,3,Part III: Expression of the Genome,This part concerned with one of the greatest challenges in understanding the genehow the gene is expressed,2019/6/30,4,Ch 12: Mechanisms of transcription Ch 13: RNA splicing Ch 14: Translation Ch 15: The genetic code,CHAPTER 7 Tran
3、slation （第七章 翻译）,2019/6/30,6,The revised central dogma,Translation,RNA processing,DNA repair and recombination,基因组的保持,基因组的表达,2019/6/30,7,What is translation?,-it is the story about decoding the genetic information contained in messenger RNA (mRNA) into proteins,genetic information: nucleotides in me
4、ssenger RNA (mRNA),protein: linear sequences of amino acids in protein,2019/6/30,8,The language of mRNA: four alphabet (A,G,C,U),The language of protein: twenty alphabet (amino acid),translation,2019/6/30,9,DNA：,5-ATGAGTAACGCG-3,3- TACTCATTGCGC-5,Nontemplate strand,template strand,transcription,mRNA
5、：,5-AUGAGUAACGCG-3,translation,Protein：,Met-Ser-Asn-Ala,2019/6/30,10,Translation extremely costs,In rapid growing bacterial cells, protein synthesis consumes 80% of the cells energy 50% of the cells dry weight,Why?,2019/6/30,11,Outline,Topic 1-4: Four components of translation machinery mRNA tRNA at
6、tachment of amino acids to tRNA (aminoacyl-tRNA synthetases) ribosome Topic 5-7: Translation process initiation; elongation; termination Topic 8: Antibiotics (抗生素）and translation,2019/6/30,12,Basic machinery of Translation,mRNAs (5% of total cellular RNA)-message RNA tRNAs (15%)-transfer RNA aminoac
7、yl-tRNA synthetases (氨酰tRNA合成酶) ribosomes (核糖体): rRNA,2019/6/30,13,What is the key attribute of each component?,How the four component work together to accomplish translation?,reductionism(还原论),holism (整体论),philosophy,2019/6/30,14,Questions addressed in “Basic machinery of Translation”,What is the o
8、rganization of nucleotide sequence information in mRNA? What is the structure of tRNAs? How do aminoacyl tRNA synthetases recognize and attach the correct amino acids to each tRNA? How does the ribosome orchestrate the translation process?,2019/6/30,15,Topic 1: mRNA(信使RNA),DNA 3-nt-long codons (三联体密
9、码子) specify the order of amino acids,mRNA,transcription,2019/6/30,16,DNA：,5-ATGAGTAACGCG-3,3- TACTCATTGCGC-5,Nontemplate strand,template strand,transcription,mRNA：,5-AUGAGUAACGCG-3,translation,Protein：,Met-Ser-Asn-Ala,2019/6/30,17,1-1 polypeptide chains are specified by ORF,open reading frame (ORF,开
10、放阅读框) : a contiguous, non-overlapping string of codons Each ORF specifies a single protein and begins with a start codon and ends with a stop codon,2019/6/30,18,The start codon （起始密码子） the first codon of an ORF In bacteria : AUG, GUG, or UUG (5-3) In eukaryotic cells: 5-AUG-3 Functions: 1.Specifies
11、the first amino acid to be incorporated into the growing polypeptide chain 2.Defines the reading frame （阅读框）for all subsequent codons,2019/6/30,19,start codon,2019/6/30,20,The stop codon （终止密码子） UAG, UGA, or UAA (5-3) Functions: 1.Defines the end of ORF 2.Signal termination of polypeptide synthesis,
12、2019/6/30,21,open reading frame (ORF): a contiguous stretch of codons “read” in a particular frame (decided by start codon) “open” to translation because it lacks a stop codon (until the last codon in ORF),2019/6/30,22,Eukaryotic mRNA (真核生物mRNA) monocistrionic, 单顺反子,polypeptide,mRNA,2019/6/30,23,Pro
13、karyotic mRNA(原核生物mRNA) polycistrionic,多顺反子,Polypeptide-1,Polypeptide-2,Polypeptide-3,Why?,ORF1,ORF2,ORF3,2019/6/30,24,Eukaryotic mRNA (monocistrionic, 单顺反子): one mRNA contains one ORF one mRNA Prokaryotic mRNA (polycistrionic,多顺反子): one mRNA contains multiple ORFs one mRNA,One ploypeptide,Multiple
14、polypeptides (related function),2019/6/30,25,1-2 Prokaryotic mRNAs have a ribosome binding site (核糖体结合位点)that recruits the translational machinery(核糖体),原核生物mRNA结构特点：,2019/6/30,26,Ribosome binding site (RBS) or SD-sequence: complementary with the sequence at the 3 end of 16S rRNA.,ORF1,ORF2,ORF3,2019
15、/6/30,27,Ribosome (核糖体): rRNA,2019/6/30,28,Translational coupling(翻译耦合),5-AUGA-3,Finding of Ribosome binding site (RBS) or SD-sequence,Scientific question: how can the cell recognize the correct start codon ?,AUG,AUG,AUG,start codon,ordinary codon,Model: R17 phage infects E. coli,R17 phage: three ge
16、nescoding for A protein, coat protein and replicase (复制酶）,Phenomenon: 生物学现象,E.coli （大肠杆菌）ribosome can translate all three phage genes in vitro(体外） Ribosome from another bacterium Bacillus （芽孢杆菌）can only translate the A protein gene,Why?,If the 30S subunit is from E.coli, phage coat protein gene can
17、be translated,If the 30S subunit is from Bacillus, phage coat protein gene can not be translated,Experimental results:,30S subunit （ 核糖体30S 小亚基）is the key factor determining the translation,?,?,30S subunit was further dissociated into RNA and protein,If the 16s rRNA is from E.coli, phage coat protei
18、n gene can be translated,If the 16s rRNA is from Bacillus, phage coat protein gene can not be translated,16s rRNA is the key factor determining the translation,J. Shine and L. Dalgarno find the SD sequence:,More powerful experimental evidence for the importance of SD-sequence,SD: GGAGG,Human growth
19、hormone gene,wild type E.coli cell (16s rRNA: CCUCC),Produce much human growth hormone protein （人生长激素蛋白）,SD: GUGUG,Human growth hormone gene,Produce no human growth hormone protein,wild type E.coli cell (16s rRNA: CCUCC),突变的研究思想,SD: GUGUG,Human growth hormone gene,Produce much human growth hormone p
20、rotein,mutated type E.coli cell (16s rRNA: CACAC),Phenomenon （现象）,Discovery of mechanism （机制） behind this phenomenon,New finding of Ribosome binding site or SD-sequence,Scientific research:,Question: how can the cell recognize the correct start codon ?,AUG,AUG,AUG,start codon,ordinary codon,AUG,AUG,
21、AUG,start codon,ordinary codon,SD-sequence,2019/6/30,41,1-3 Eukaryotic mRNA are modified at their 5 and 3 ends to facilitate translation,polypeptide,mRNA,戴帽穿鞋,2019/6/30,42,RNA processing： 5 end capping,The “cap”: a methylated guanine joined to the RNA transcript by a 5-5 linkage (甲基化的鸟嘌呤）,2019/6/30,
22、43,Once,Kozak sequence,Eukaryotic mRNA uses a methylated cap to recruit the ribosome. Once bound, the ribosome scans the mRNA in a 5-3 direction to find the AUG start codon-scanning(扫描),2019/6/30,44,Kozak sequence：5-G/ANNAUGG-3,+1,-3,+4,How was the Kozak sequence found?,How can we prove that the Koz
23、ak sequence is important to translation?,Question:,How was the Kozak sequence found?,Phenomenon,Raise question,New finding of Kozak sequence,AUG,AUG,AUG,start codon,5cap,ribosome,scanning,AUG,AUG,AUG,start codon,5cap,ribosome,scanning,Kozak sequence：5-G/ANNAUGG-3,start codon,+1,-3,+4,Next question:,
24、Is the Kozak sequence really important to translation initiation? How can we prove that the Kozak sequence is important to translation?,Experimental evidence for proving the importance of the Kozak sequence:,Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates t
25、ranslation by eukaryotic ribosomes. Cell,1986,44:283-292,研究论文（原始研究工作）的学习,2019/6/30,49,不仅仅知道分子生物学知识是什么？而且也了解分子生物学知识是怎么得来的？有哪些实验证据支持所学的基本理论和概念？ 不仅仅学习基本知识和概念，也学习科学研究的方法，接受科学思维的训练 从别人的研究中获得启发，获得新的IDEA 加深对基本概念的理解,为什么要学习研究论文的研究工作？,Idea of mutation (genetic method),Kozak sequence：5-G/ANNAUGG-3,start codon,
26、+1,-3,+4,Mutated to other nucleotide,Detect the effect of mutation on translation efficiency,研究思路：,Design of the experiment,5-G/ANNATGG,+1,-3,+4,Model ：rat preproinsulin gene(小鼠前胰岛素原基因）,rat preproinsulin gene,COS cells growing in medium containing 35S-Methionine(甲硫氨酸）,Detect the amount of preproinsu
27、lin protein(35S labeled),Introduce into COS cell,5-G/ANNATGG,+1,-3,+4,rat preproinsulin gene,mutant,+2,+3,+1,protein,G/ANNATGG,+1,-3,+4,rat preproinsulin gene,G/ATTATGG,start codon,A,T,G,A,T,G,A,T,G,A,T,G,A,T,G,A,T,G,A,T,G,A,T,G,+1,-3,+4,C,T,G,protein,2019/6/30,54,The Kozak sequence is really import
28、ant to translation initiation,Conclusion:,Poly-A in the 3 end promotes the efficient recycling of ribosomes, thus increasing the translation efficiency,2019/6/30,56,Basic concepts:,ORF: start codon, stop codon, monocistrionic, polycistrionic Prokaryotic mRNA : ribosome binding site (核糖体结合位点) Eukaryo
29、tic mRNA: a methylated cap to recruit the ribosome, scanning, Kozak sequence,2019/6/30,57,Topic 2: tRNA,The genetic information in mRNA cannot be recognized by amino acids directly (the chain group of amino acid cannot interact with base group of mRNA). The genetic code has to be recognized by an ad
30、aptor molecule (transfer RNA), and this adaptor has to accurately recruit the corresponding amino acid.,2019/6/30,58,tRNA function,Nucleotide codon,amino acid,adaptor(转配器),tRNA,The story of finding tRNA,Cricks hypothesis: Adaptor molecular Can recognize both nucleotide and amino acid A type of small
31、 RNA of unknown function,The research work of Zamecnik in 1957,2019/6/30,61,The research work of Zamecnik in 1957,Model: a cell-free protein synthesis system from rat,One of the components of this system was a so-called pH5 enzyme fraction that can direct the mRNA translation,Most of pH5 enzyme frac
32、tion were proteins, but Zamecnik discovered that this mixture also included a small RNA,This RNA could be coupled to amino acid,Mix the small RNA with the pH5 enzyme, ATP and 14Cleucine More labeled leucine added to the mixture, the more was attached to the RNA,14Cleucine,14CRNA,A perfect correspond
33、ence between the loss of radioactive leucine from RNA and gain of the leucine by the microsome protein Incorporation of leucine from leucyle-tRNA into protein on ribosome,Mixing the 14Cleucine-charged RNA with microsome ( containing ribosome),14C 放射性强度,时间,2019/6/30,66,Why nature select tRNA as the a
34、daptor molecule?,Watson-Crick principle: base pairing,2019/6/30,67,Each tRNA is attached to a specific amino acids and each recognizes a particular codon in the mRNA,2-1: tRNA are adaptors between codons and amino acids,2019/6/30,68,1. tRNAs are 75-95 nt in length. 2. There are many modified bases(修
35、饰碱基), which sometimes accounting for 20% of the total bases in one tRNA molecule.,Primary structure,2019/6/30,69,Fig 14-3 unusual bases,Pseudouridine (U，假尿嘧啶) is a modified base. These modified bases in tRNA lead to improved tRNA function,2019/6/30,70,form four stems (arms) and three loops Self-comp
36、lementary regions within tRNA,2-2: tRNAs share a common secondary structure that resembles a cloverleaf(三叶草型),2019/6/30,71,The site of attachment of amino acid,A three-nucleotide-long sequence responsible for recognizing the codon by base pairing with mRNA,cloverleaf structure of tRNA,2019/6/30,72,2
37、019/6/30,73,2-3: tRNAs have an L-shaped 3-D structure,Fig 14-5 the 3-D structure of tRNA,2019/6/30,74,The anticodon loop at one end and the amino acid acceptor site at the other. The base pairing is strengthened by base stacking interactions.,2019/6/30,75,Topic 3: attachment of amino acids to tRNA,A
38、mino acids should be attached to tRNA first before adding to polypeptide chain. Charged tRNA (负载tRNA) Uncharged tRNA (空载tRNA),2019/6/30,76,3-1 tRNAs are charged by attachment of an amino acid to the 3 terminal A of the tRNA via a high energy acyl linkage(高能酰基),2019/6/30,77,Aminoacyl tRNA synthetase
39、(氨酰tRNA合成酶)catalyze this reaction: has three binding sites for ATP, amino acid and tRNA,2019/6/30,78,3-2 Aminoacyl tRNA synthetases charge tRNA in two steps,Adenylylation (腺苷酰化) of amino acids: transfer of AMP to the COO- end of the amino acids. 2. tRNA charging: transfer of the adenylylated amino a
40、cids to the 3 end of tRNA, generating aminoacyl-tRNAs (氨酰tRNA).,2019/6/30,79,Step 1-Adenylylation of amino acids(腺苷酰化氨基酸）,腺苷酰化氨基酸,2019/6/30,80,Step 2-Transfer of the adenylated amino acid to tRNA,aminoacyl-tRNAs(氨酰tRNA),tRNAGlu,2019/6/30,81,3-3： each aminoacyl tRNA synthetase attaches a single amino
41、 acids to one or more tRNAs,Each of the 20 amino acids is attached to the appropriate tRNA (s) by aminoacyl-tRNA synthetases. Most amino acids are specified by more than one codon, and by more than one tRNA as well (isoaccepting tRNAs,同工tRNA).,2019/6/30,82,The specificity determinants for accurate r
42、ecognition are clusters at two distinct sites: the acceptor stem and the anti-codon loop.,2019/6/30,83,鉴别碱基,2019/6/30,84,3-4 Aminoacyl-tRNA formation is very accurate: selection of the correct amino acid(识别正确的氨基酸),tRNA,aminoacyl tRNA synthetase,amino acid,Why the aminoacyl tRNA synthetase must recog
43、nize the amino acid and tRNA with very high accuracy and fidelity?,Question:,tRNA,aminoacyl tRNA synthetase,amino acid,2019/6/30,86,3-5 Ribosomes is unable to discriminate between correctly or incorrectly charged tRNAs (核糖体不能够识别是否携带正确的氨基酸),The ribosome “blindly” accepts any charged tRNA that exhibit
44、s proper codon-anticodon interaction, whether or not the tRNA is charged with the correct amino acid,2019/6/30,87,One classic biochemical experiment,半胱氨酰tRNA,丙氨酰tRNA,半胱氨酸反密码子,半胱氨酸反密码子,2019/6/30,88,mutation,2019/6/30,89,The translation machinery relies on the high fidelity (高保真）of aminoacyl tRNA synt
45、hetase to ensure the accurate decoding of mRNA,2019/6/30,90,Topic 4: the ribosome,The ribosome is the macromolecular machine that directs the synthesis of proteins The ribosome is composed of at least three RNA moleculars and more than 50 different proteins.,2019/6/30,91,翻译的基本装置之一：进行蛋白质翻译的的重要细胞器，合成蛋
46、白质的车间 2009年来自英国，美国和以色列的三位科学家正是因为“对核糖体的结构和功能的研究”而获得诺贝尔化学奖,2019/6/30,98,Coupling of transcription and translation in prokaryotes,ribosome,2019/6/30,99,4-1 the ribosome is composed of a large and a small subunit,2019/6/30,100,2019/6/30,101,2019/6/30,102,The large subunit contains the peptidyl transfera
47、se center(肽基转移酶中心), which is responsible for the formation of peptide bonds. The small subunit interacting with mRNA contains the decoding center(解码中心), in which charged tRNAs read or “decode” the codon units of the mRNA.,2019/6/30,103,4-2: the large and the small subunits undergone association and
48、dissociation during each cycle of translation.,2019/6/30,104,Ribosome cycles (核糖体循环): The small and large ribosome subunits associate with each other and translate the mRNA, then dissociate after each round of translation,2019/6/30,105,Overview of the events of ribosome cycle,mRNA/initiator tRNA/ sm
49、all ribosomal subunit,polypeptide,2019/6/30,106,4-3 New amino acids are attached to the C-terminus of the growing polypeptide chain.,Protein is synthesized in a N- to C- terminal direction,2019/6/30,107,4-4 Peptide bonds(肽键)are formed by transfer of the growing peptide chain from peptidyl- tRNA(肽酰tRNA) to aminoa